Use of γ-tocopherol and its oxidative metabolite LLU-α in the treatment of disease

ABSTRACT

The present invention is generally related to the discovery of the therapeutic benefit of administering γ-tocopherol and γ-tocopherol derivatives. More specifically, the use of γ-tocopherol and racemic LLU-α, (S)-LLU-α, or γ-tocopherol derivatives as antioxidants and nitrogen oxide scavengers which treat and prevent high blood pressure, thromboembolic disease, cardiovascular disease, cancer, natriuretic disease, the formation of neuropathological lesions, and a reduced immune system response are disclosed.

I. CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to continuation U.S. patent applicationSer. No. 09/461,645, filed Dec. 14, 1999, now U.S. Pat. No. 6,242,479which is a continuation application of U.S. patent application Ser. No.09/215,608, filed Dec. 17, 1998, now U.S. Pat. No. 6,048,891 which arehereby expressly incorporated by reference in their entireties.

II. FIELD OF INVENTION

The present invention is generally related to the discovery of thetherapeutic benefit of administering γ-tocopherol and γ-tocopherolderivatives. More specifically, the use of γ-tocopherol and racemicLLU-α, (S)-LLU-α, or other γ-tocopherol derivatives as antioxidants andnitrogen oxide scavengers which treat and prevent high blood pressure,thromboembolic disease, cardiovascular disease, cancer, natriureticdisease, the formation of neuropathological lesions, and a reducedimmune system response are disclosed.

III. BACKGROUND OF THE INVENTION

Vitamin E, an essential fat-soluble vitamin, encompasses eight naturallyoccurring compounds in two classes. The first class, tocopherols, havefour members designated alpha, beta, gamma and delta. The two majorforms, α-tocopherol and γ-tocopherol, differ structurally only by amethyl group substitution at the 5-position. The second class,tocotrienols, are molecules related to the tocopherols and also consistof four members designated alpha, beta, gamma and delta. The tocotrienolstructure differs from the tocopherols by possessing three double bondsin their side chain rather than being saturated.

One of the important chemical features of the tocopherols is that theyare redox agents which act under certain circumstances as antioxidants.In acting as an antioxidant, tocopherols presumably prevent theformation of toxic oxidation products, such as perioxidation productsformed from unsaturated fatty acids. Early on, investigators attributedmost if not all of the biological activity of the tocopherols to theirability to act as antioxidants. More recently, however, other biologicalactivities have been associated with tocopherols including themodulation of signal transduction, modulation of phospholipidmetabolism, inhibition of protein kinase C, inhibition of phospholipaseA and inhibition of prostaglandin production. (Meydani and Mosen, TheLancet 345(8943):170-175 (1995)).

Further, it has recently been discovered that individual members in theclass of tocopherols may exhibit different biological properties fromone another despite their structural similarity. Some investigators, forexample, believe that γ-tocopherol, unlike α-tocopherol, acts in vivo asa trap for membrane-soluble electrophilic nitrogen oxides and otherelectrophilic mutagens. (Christen et al. Proc. Natl. Acad. Sci. 94:3217-3222 (1997)). In contrast, others report that α-tocopherol is amore powerful antioxidant and has ten times the biological activity ofγ-tocopherol. (Meydani and Mosen, The Lancet 345(8943):170-175 (1995)).Alpha-tocopherol is also thought to be retained in the body longer thanγ-tocopherol and has been shown to preferentially reincorporate intonascent very low-density lipoproteins (LDL). (Christen et al. Proc.Natl. Acad. Sci. 94: 3217-3222 (1997)). At present, an understanding ofthe differences in biological activity of the four tocopherols and theireffect on the body is in its infancy.

Alpha tocopherol is largely considered the most important member of theclass of tocopherols because it constitutes about 90% of the tocopherolsfound in animal tissues and displays the greatest biological activity inthe commonly used bioassay systems. In consequence, vitamin Esupplements are almost exclusively made of α-tocopherol and littleinvestigation into the efficacy of supplementation with γ-tocopherol hasbeen conducted.

The therapeutic benefits of vitamin E supplementation remains a subjectof considerable debate. Several studies have proposed that vitamin Esupplementation may prevent a plethora of ills but many of these studiesfail to provide causal connections between vitamer supplementation andtherapeutic benefit; they merely indicate that a high dietary or plasmaconcentration and supplemental intake of vitamin E is associated with areduced risk of disease. In fact, some studies have failed todemonstrate that tocopherol supplementation provides any protection fromdisease. (Meydani and Mosen, The Lancet 345(8943):170-175 (1995) and(Christen et al. Proc. Natl. Acad. Sci. 94: 3217-3222 (1997)). Areliable method to treat and prevent diseases associated with oxidativestress and vitamin E deficiency is highly desirable.

SUMMARY OF THE INVENTION

The present invention reveals the discovery of the therapeutic benefitof administering γ-tocopherol and γ-tocopherol derivatives such asLLU-α. The novel use of γ-tocopherol and γ-tocopherol derivatives asantioxidants and nitrogen oxide scavengers which treat and prevent highblood pressure, thromboembolic disease, cardiovascular disease, cancer,natriuretic disease, the formation of neuropathological lesions, and areduced immune system response are disclosed.

One embodiment of the present invention is a medicament comprisingγ-tocopherol and LLU-α with and without additional active ingredientsthat are effective in producing a natriuretic effect. Another embodimentis a medicament comprising γ-tocopherol, α-tocopherol, and LLU-α withand without additional active ingredients that are effective inproducing a natriuretic effect. A further embodiment is a medicamentcomprising γ-tocopherol, β-tocopherol, and LLU-α, with and withoutadditional active ingredients that are effective in producing anatriuretic effect. Still further, an embodiment comprisingα-tocopherol, γ-tocopherol, β-tocopherol, and LLU-α, with and withoutadditional active ingredients that are effective in producing anatriuretic effect, is disclosed. In the alternative, the embodimentsdescribed above may include (S)-LLU-α or other γ-tocopherol derivativesinstead of LLU-α.

According to the methods of treatment and prevention disclosed, themedicaments described above are administered to subjects suffering fromhigh blood pressure, thromboembolic disease, atherosclerosis,cardiovascular disease, cancer, natriuretic disease, the formation ofneuropathological lesions, and a reduced immune system response. Onemethod involves the administration of a therapeutically beneficialamount of γ-tocopherol, with or without supplementation of LLU-α, tosubjects suffering from a high blood pressure so as to treat and preventthis condition. By another method, a therapeutically beneficial amountof γ-tocopherol, with or without supplementation of LLU-α, isadministered to treat and prevent thromboembolic disease. A relatedmethod to treat and prevent the aggregation of platelets and/or bindingof platelets to adhesive proteins is also disclosed.

Another method contemplated by the present inventor involves theadministration of a therapeutically beneficial amount of γ-tocopherol,with or without supplementation of LLU-α, to treat and preventcardiovascular diseases, such as ischemia, angina, edematous conditions,artherosclerosis, LDL oxidation, adhesion of monocytes to endothelialcells, foam-cell formation, fatty-streak development, plateletadherence, platelet aggregation, smooth muscle cell proliferation, andreperfusion injury. Further, a method to treat and prevent cancers, suchas lung cancer, prostate cancer, breast cancer, and colon cancer byadministering a therapeutically beneficial amount of γ-tocopherol, withor without supplementation of LLU-α are presented.

Methods of treatment and prevention of natriuretic diseases, such ashypertension, high blood pressure, ischemia, angina pectoris, congestiveheart failure, cirrhosis of the liver, nephrotic syndrome, ineffectiverenal perfusion, or ineffective glomerular filtration, by administeringa therapeutically beneficial amount of γ-tocopherol, with or withoutsupplementation of LLU-α are also provided. Additionally, methods oftreating and preventing neurological diseases including hyporeflexia,opthalmoplegia, and axonal dystrophy using a therapeutically beneficialamount of γ-tocopherol, with or without supplementation of LLU-α, aredescribed. Finally, methods to improve a subject's immune systemresponse and a related method to reduce the production of free-radicalsby administering a therapeutically beneficial amount of γ-tocopherol,with or without supplementation of LLU-α, is revealed.

CHEMICAL STRUCTURE OF LLU-α

FORMULA A shows the structural formula of LLU-α.

FORMULA B shows the structural formula of (S)-LLU-α.

VI. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, a novel method for thetreatment and prevention of high blood pressure, thromboembolic disease,atherosclerosis, cancer, natriuretic disease, the formation ofneuropathological lesions, and a reduced immune system response isprovided. The method involves administering orally or parenterallysubstantially pure γ-tocopherol or a formulation comprising γ-tocopheroland racemic LLU-α, (S)-LLU-α, or other γ-tocopherol derivative.

By “LLU-α” is meant the compound6-hydroxy-2,7,8-trimethylchroman-2-propanoic acid, molecular weight of264.1362 and molecular formula of C₁₅H₂₀O₄. LLU-α may be in the racemicform or as the S enatiomer (also denoted as (S)-LLU-α). A generaldiscussion of the isolation and characterization of LLU-α is provided byWechter et al. (U.S. patent application Ser. No. 08/290,430) thedisclosure of which is hereby incorporated by reference.

By “γ-tocopherol derivative” is meant γ-tocopherol metabolites andsynthetic chroman derivatives including, but not limited to, LLU-α,LLU-γ, racemic chromans, chroman methyl esters, chroman esters, chromanamides, R₄ chroman esters, oxidized chroman derivatives, racemic2,5,7,8-tetramethyl-2-(β-carboxyethyl)-6-hydroxy chroman,2,5,7,8-tetramethyl-2-(β-carboxyethyl)-chroman,2,7,8-trimethyl-2-(β-carboxyethyl) chroman, racemic4-methyl-6-(5,6-dimethylbenzohinoyl)-4-hexanolid,4-Methyl-6-(3,5,6-trimethylbenzochinoyl)-4-hexanolid,(S)-4-Methyl-6-(5,6-dimethylbenzochinoyl)-4-hexanolid,2,7,8-Trimethyl-2-(β-carboxyethyl)-6-acetyl chroman,2,7,8-Trimethyl-2-(β-carboxyethyl)-6-acetyl chroman methyl ester, andbenzodipyran methyl ester. Many γ-tocopherol derivatives are natriureticcompounds but the meanining of “γ-tocopherol derivative” is not intendedto be limited to only natriuretic compounds. Other γ-tocopherolmetabolites and synthetic chroman derivatives may be known by those ofskill in the art or will be discovered in the future and are encompassedby this definition.

By “natriuretic disease” is meant diseases associated with abnormalexcretion of sodium from the body. The term natriuretic disease includesbut is not limited to hypertension, high blood pressure, ischemia,angina pectoris, congestive heart failure, cirrhosis of the liver,nephrotic syndrome, ineffective renal perfusion, and ineffectiveglomerular filtration, or any combination thereof. Other forms ofnatriuretic disease will be apparent to those of skill in the art andare encompassed by the definition as used in this invention.

As used herein, the term “natriuretic compound” refers to a compoundwhich increases the rate of sodium excretion without contributing tosignificant potassium loss in a mammal upon administering the compoundto the mammal. The term “natriuretic compound” also refers to both thenative compound and in vitro or in vivo modifications which retainnatriuretic activity. It is understood that limited modifications,substitution or deletions of functional groups may be made withoutdestroying the biological activity. Moreover, it will be recognized bythose skilled in the arts of chemistry and pharmaceutical preparationthat many derivatives can be made which are biologically and chemicallyequivalent to, or even more active than, the indicated compoundshereinafter. Examples of equivalent compounds include esters, ethers,amides and salts of the foregoing compounds.

“Substantially purified,” when used to describe the state of thenatriuretic compound, denotes the compounds essentially free ofproteins, steroids, and other material normally associated or occurringwith natriuretic compounds in its native environment.

As used herein, the term “post salt peak” refers to material eluted froma G-25 Sephadex column which appears immediately after the sodium,potassium, urea and creatinine containing fractions which has uv.absorbance at 290 nm.

A material is “biologically active” if it is capable of increasingnatriuresis in an in vivo assay as described herein.

By “thromboembolic disease” is meant diseases characterized by plateletaggregation, platelet adhesion to adhesive proteins, or platelethyperactivity. Although thromboembolic disease is commonly associated ininsulin-dependent diabetic patients, this understanding is not intendedto limit the invention. Elderly patients and patients suffering fromvarious forms of cardiovascular disease exhibit platelet aggregation,platelet adhesion to adhesive proteins, and platelet hyperactivity whichcan be defined as forms of thromboembolic disease for the purposes ofthis invention. Other forms of thromboembolic disease will be apparentto those of skill in the art and are encompassed by the definition usedin this invention.

By “cardiovascular disease” is meant diseases associated with thecardiopulmonary and circulatory systems including but not limited toischemia, angina, edematous conditions, artherosclerosis, LDL oxidation,adhesion of monocytes to endothelial cells, foam-cell formation,fatty-streak development, platelet adherence, and aggregation, smoothmuscle cell proliferation, reperfusion injury, and other conditionsknown by those of skill in the art to be related to the pathogenesis ofcardiovascular disease.

By “cancer” is meant diseases that have been associated withmutagenesis, cell transformation, oncogenesis, neoplasia, or metastasis,including but not limited to, various forms of lung cancer, prostatecancer, breast cancer, and colon cancer, or any combination thereof.Other forms of cancer will be apparent to those of skill in the art andare encompassed by the definition used in this invention.

By “neurological disease” is meant diseases associated with the brainand nervous system, including but not limited to, hyporeflexia,proprioception, opthalmoplegia, and axonal dystrophy. Other forms ofneurological diseases will be apparent to those of skill in the art andare encompassed by the definition as used in this invention.

Gamma-tocopherol is a water-insoluble, non swelling amphiphile, as aretriglycerides and cholesterol. Thus, many of the processes involved inthe absorption of lipids are also required for absorption ofγ-tocopherol such as emulsification, solubilization within mixed bilesalt micelles, uptake by the small intestine, packaging withinlipoprotein particles, and secretion into the circulation via thelymphatic system. Gamma-tocopherol is transferred to tissues in much thesame manner as other lipids and spontaneous transfer and exchange oftocopherol between cell membranes has been documented. Sinceγ-tocopherol is rapidly absorbed in the lipids of various tissuesincluding the liver, its antioxidant and radical scavenger activitiesprimarily occur in the lipid phase and only tangentially in the aqueousphase. LLU-α, on the other hand, is considerably more hydrophilic thanγ-tocopherol and acts as an antioxidant, a natriuretic compound, andradical scavenger in primarily the aqueous phase. Thus, the presentinventor contemplates a method to treat and prevent disease whichemploys supplements comprising γ-tocopherol with and withoutfortification with racemic LLU-α, (S)-LLU-α, or other γ-tocopherolderivative so as to selectively provide natriuretic redox agents to thelipid and aqueous phases of a patient's body.

The preparation of soft gelatin capsules comprising commerciallyavailable γ-tocopherol in doses of 200 to 800 mg is understood by thoseof skill in the art. The γ-tocopherol may be present as the free alcoholor the acetate or succinate ester. A supplement of γ-tocopherolpreferably contains at least 60-65% (weight to weight) γ-tocopherol andup to 10% α-tocopherol and 25% β-tocopherol as isolated from soy oil, orin certain circumstances up to 25% δ-tocopherol. Particularly preferredcompositions include at least 70% γ-tocopherol. These formulations areonly intended to guide one of skill in the art and formulations ofγ-tocopherol that would be effective for use in the disclosed methodsmay include as low as 50% (weight to weight) γ-tocopherol or up to 100%(weight to weight) γ-tocopherol, but desirably contain 55% (weight toweight) γ-tocopherol to 95% (weight to weight) γ-tocopherol.

In another embodiment of this invention, soft gelatin capsulescomprising commercially available γ-tocopherol are fortified with anatriuretic compound such as LLU-α, (S) LLU-α, or other γ-tocopherolderivative some of which may be present as the free acid or a simpleester. One aspect of the invention, for example, comprises a natriureticcompound having the formula I:

in which

R is O, S, SO, SO₂, a secondary or tertiary amine group, a phosphategroup, a phosphoester group, or an unsubstituted or substitutedmethylene group,

R₁ and R₂ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic or aromatic ring,

R₃ and R₄ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic, aromatic or heterocyclic ring,

R₅ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester orunsubstituted or substituted amine,

R₆ is COOH, COOR₇, CONH₂, CONHR₇, CONR₇R₈, NH₂, NHR₇, NR₇R₈, or acarboxylate salt,

R₇ and R₈ independently are unsubstituted or substituted alkyl, aryl,alkaryl, aralkyl, alkenyl or alkynyl,

n is 0 to 3, and

m is 0 to 5.

As used herein, the term “substituted” denotes the presence of one ormore substituent such as alkyl, aryl, alkaryl, aralkyl, ether orhalogen. More particular substituents include C₁₋₆ unbranched orbranched alkyl, such as methyl, ethyl, propyl, n-butyl, sec-butyl andtert-butyl, and C₆₋₁₂ aryl, particularly phenyl.

In a preferred embodiment, R is O. Also preferably, n=1. Preferably,m=2.

R₆ preferably is COOH.

Preferably, R₃ is H or OH. Also preferably, R₄ is H or CH₃.

In a preferred embodiment, R₁, R₂ and R₅ are CH₃.

Exemplary preferred compounds of formula I include those in which R isO, R₁, R₂ and R₅ are CH₃, R₃ is OH, R₄ is H or CH₃, R₆ is COOH, n=1 andm=2.

Other exemplary preferred compounds of formula I includes those in whichR is O, R₁, R₂ and R₅ are CH₃, R₃ is H, R₄ is H or CH₃, R₆ is COOH, n=1and m=2.

In a preferred embodiment, R₇ is a C₁₋₆ alkyl group, in particular CH₃.

In another preferred embodiment, R₃ is OH.

Compounds used in the present invention can also be obtained bymodifying the above recited formula in numerous ways while preservingnatriuretic activity. Examples of such active derivatives includecompounds of formulae II-V, below.

In all formulae described herein, moieties having like designations areconsidered to correspond to each other as like moieties in relatedcompounds.

Another aspect of the invention comprises natriuretic compounds havingthe formula II:

wherein

R₁ and R₂ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic or aromatic ring,

R₃ and R₄ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic, aromatic or heterocyclic ring,

R₅ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester orunsubstituted or substituted amine,

R₆ is COOH, COOR₇, CONH₂, CONHR₇, CONR₇R₈, NH₂, NHR₇, NR₇R₈, or acarboxylate salt,

R₇ and R₈ independently are unsubstituted or substituted alkyl, aryl,alkaryl, aralkyl, alkenyl or alkynyl,

R₉ is hydroxyl or unsubstituted or substituted alkoxyl,

n is 0 to 3, and

m is 0 to 5.

In a preferred embodiment, R₁, R₂ and R₅ are CH₃. Preferably, R₃ is OH.

R₄ preferably is H.

Additionally, it is preferred that n=1. Preferably m=2.

In a preferred embodiment, R₆ is COOCH₂CH₃ and r₉ is OH. In anotherpreferred embodiment, R₆ is COOH and R₉ is CH₃CH₂O.

Specific examples includes the following:

A further aspect of the invention comprises natriuretic compounds havingthe formula III:

wherein

R₁ and R₂ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic or aromatic ring,

R₃ and R₄ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic, aromatic or heterocyclic ring,

R₅ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester orunsubstituted or substituted amine,

n is 0 to 3, and

q is 0 to 4.

In preferred embodiments, n=1. Also preferred are compounds in whichm=2.

Exemplary natriuretic compounds of formula III include the following:

The instant invention comprises other natriuretic compounds having theformula IV:

wherein

R₁ and R₂ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic or aromatic ring,

R₄ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester,unsubstituted or substituted amine, amide, halogen or unsubstituted orsubstituted sulfonyl,

R₅ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester orunsubstituted or substituted amine,

R₆ is COOH, COOR₇, CONH₂, CONHR₇, CONR₇R₈, NH₂, NHR₇, NR₇R₈, or acarboxylate salt,

R₇ and R₈ independently are unsubstituted or substituted alkyl, aryl,alkaryl, aralkyl, alkenyl or alkynyl,

R₉ is hydroxyl or unsubstituted or substituted alkoxyl,

n is 0 to 3, and

m is 0 to 5.

Preferably n=1. Also, preferably m=2.

Specific compounds of the invention according to formula IV include:

Natriuretic compounds of formula V are also combined with γ-tocopherolto make the medicaments of the instant invention:

wherein

R₁ and R₂ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic or aromatic ring,

R₄ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester,unsubstituted or substituted amine, amide, halogen or unsubstituted orsubstituted sulfonyl,

R₅ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester orunsubstituted or substituted amine,

n is 0 to 3, and

q is 0 to 4.

Preferred embodiments are those in which n=1. Also, it is preferred thatm=2.

Included in the inventive compounds of formula V are:

In accordance with another aspect of present invention, medicamentshaving the formula Ia and γ-tocopherol are contemplated.

in which

R is O, S, SO, SO₂, a secondary or tertiary amine group, a phosphategroup, a phosphoester group, or an unsubstituted or substitutedmethylene group,

R₁ and R₂ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic or aromatic ring,

R₃ and R₄ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic, aromatic or heterocyclic ring,

R₅ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester orunsubstituted or substituted amine,

R₆ is COOH, COOR₇, CONH₂, CONHR₇, CONR₇R₈, NH₂, NHR₇, NR₇R₈, or acarboxylate salt,

R₇ and R₈ independently are unsubstituted or substituted alkyl, aryl,alkaryl, aralkyl, alkenyl or alkynyl,

n is 0 to 3, and

m is 0 to 5.

In preferred embodiments,

(i) when R is O, R₁, R₂ and R₅ are CH₃, R₃ and R₆ are OH, and R₄ is H,m=2 to 5;

(ii) when R is O R₁ is H or CH₃, R₂ is H, CH₃, C(CH₃)₃ or CH(CH₃)₂, R₃is OH or CH₃COO, R₄ is CH₃ or CH(CH₃)₂, R is H, CH₃or CH₂CH₃, and R₆ isH, OH, OCH₃, OCH₂CH₃ or NH₂, m=1 to 5;

(iii) when R is O, R₁ and R₅ are CH₃, R₂ and R₄ are H, R₃ is OH orCH₃COO, and R₆ is OH or CH₃O, m is not 2;

(iv) when R is O, R₁, R₂ and R₅ are CH₃, R₃ is OH or CH₃COO, R₄ is alkylhaving at least two carbon atoms, and R₆ is H, OH or ester, m=1; and

(v) when R₁, R₂ and R₅ are methyl, R₃ and R₆ are OH and R₄ is alkyl,m=2.

Certain medicaments of the present invention comprise natriureticcompounds that have been isolated in substantially pure form. Thenatriuretic compounds are obtained from a variety of sources, includingurine, hypothalamus, adrenal, liver, kidney, plasma, blood and culturedcells. Human uremic urine is the preferred source, although normal humanurine or hypertensive human urine may also be used.

One of the isolated natriuretic compounds used to make a medicament ofthe present invention is LLU-α. (See FIGS. 1 and 2). LLU-α has thefollowing properties: a major ultraviolet absorbance peak at about 210nm; a broad secondary peak at about 295 nm; instability in dilute base;capability of esterification by reaction with CH₂N₂. The compound iscapable of increasing sodium excretion in the urine in mammals without acorresponding increase in potassium excretion, and does not cause asignificant change in mean arterial pressure. The compound additionallyacts as a cardio-selective free radical scavenger.

Medicaments of the instant invention also comprise another isolatednatriuretic compound, named LLU-γ, which has the following properties: amajor ultraviolet absorbance peak at about 220 nm; a secondary peak atabout 268 nm; high instability in the presence of O₂ or in dilute base.It is capable of increasing sodium excretion in mammalian urine withouta corresponding increase in potassium excretion, although potassiumexcretion (kaliuresis) may be observed occasionally after infusion ofthe compound into conscious rats. In addition, it does not cause asignificant change in mean arterial pressure and it shows no inhibitionof the sodium pump.

Natriuretic compounds which comprise the present invention can bepurified by a number of methods, particularly those exemplified herein.In a preferred method within the invention, collected urine is processedby ultrafiltration (≦3 kDa), gel filtration chromatography (G-25) andextraction with isopropanol and diethyl ether. The organic solublematerial is then subjected to sequential high-performance liquidchromatography, while assaying for the natriuretic, activity in vivo.Alternatively, collected urine is extracted with ether, separated byhigh performance liquid chromatography, and fractions are assayed fornatriuretic activity.

In a further alternative embodiment, the natriuretic compounds in themedicaments of the present invention can be synthesized using methodsknown to those skilled in the art. One such method is the methoddescribed by J. Weichet et al., Czech. Chem. Commun. 24, 1689-1694(1959), the disclosure of which is hereby incorporated by reference.This method can readily be adapted by one of ordinary skill in the artto provide a method of synthesizing the compounds of the presentinvention. Other methods to synthesize the natriuretic compounds of thepresent invention are disclosed in Wechter et al., Proc. Natl. Acad.Sci. USA 93:6002-6007 (1996) and Kantoci et al., J. Pharmacology andExperimental Therapeutics 282:648-656 (1997) which are herebyincorporated by reference.

A preferred synthetic method includes the step of reacting a compound ofthe formula VI:

in which

R₁ and R₂ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl. or jointly complete a5- or 6-member aliphatic or aromatic ring, and

R₃ and R₄ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic, aromatic or heterocyclic ring,

with a vinyl lactone of the formula VII:

in which

R₅ is H, alkyl, aryl, alkenyl, alkynyl, aromatic or ester,

n is 0 to 3, and

q is 0 to 4.

In a preferred embodiment of the foregoing synthesis, R₃ is OH.Preferably, R₄ is not simultaneously OH. A preferred compound of formulaVI is a hydroquinone, for example 2,3-dimethyl-1,4-hydroquinone.

A preferred vinyl lactone of formula VII isγ-methyl-γ-vinylbutyrolactone (R₅=CH₃, n=1, q=1).

In carrying out the foregoing reaction, preferably a catalyst is used,such as a metallic or non-metallic salt. Specific types of catalystinclude non-metallic salts which form complexes with a solvent,particularly a catalyst such as boron trifluoride diethyl etherate.

In carrying out the foregoing reaction, preferably an aprotic or proticsolvent is employed, in particular an aprotic solvent such as dioxane.The catalyst and/or the vinyl lactone is preferably diluted in theselected solvent.

Preferably the synthesis is carried out at an elevated temperature, suchas 100-110° C.

In a preferred embodiment, the foregoing reaction mixture is dilutedwith an aprotic or protic solvent, particularly an aprotic solvent suchas diethyl ether.

The desired product preferably is obtained from concentrated supernatantwhich is purified, for example, using an RP-HPLC column or silica gel.Preferred eluents for RP-HPLC include mixtures of water, acetonitrileand acetic acid. Preferred solvents for silica gel include ethyl acetateand hexane. Other purification methods, such as crystallization, can beused. Also, other eluents, such as hexane and dimethyl ketone, can beemployed.

The foregoing synthesis produces a racemic mixture, of which typicallyone enantiomer is active while the second enantiomer is less active orinactive. The racemate can be employed in compositions according to theinvention, with adjustment of the quantity to account for the presenceof the inactive enantiomer. Alternatively, the racemate can be resolvedusing conventional methods, and the active enantiomer identified andutilized. All enantiomeric forms of the compounds described herein arespecifically contemplated as being within the scope of the instantinvention.

As a byproduct of the foregoing synthesis, derivative compounds offormula VIII are produced:

These compounds can also be employed as natriuretic compounds whichcomprise the medicamnet according to the instant invention. Exemplarycompounds of formula VIII include the following benzodipyranderivatives:

All stereoisomeric forms of the foregoing compounds, including mesocompounds and diastereomeric pairs, are specifically contemplated asbeing within the scope of the instant invention.

Di-oxidized and/or di-hydrated derivatives of the compounds of formulaVIII can be obtained in a manner analogous to those used to obtaincompounds of formulae II-V from the compounds of formula I.

As mentioned previously, natriuretic compounds which comprise themedicamnets of the instant invention can be modified by formation ofesters, amides, etc. Esterification can be carried out, for example, byreaction with a solution of a diazoalkane, or with an anhydride or anacyl chloride. Amides can be formed by reaction with ammonia or anamine.

Natriuretic compounds of formulae II-V can be derived from thecorresponding natriuretic compounds produced by the foregoing method,for example, by oxidation. In a preferred embodiment of this process,when R₄═H, R₅ is not CH₃.

A preferred oxidizer for the foregoing method is a solution of ferricchloride. Other oxidants, such as KMnO₄, SeO₂, CrO₃, H₂O₂,m-chloroperbenzoic acid, Caro acid, OsO₄, HIO₄, potassium ferricyanide,silver chromate or sodium perborate, can also be used.

Scheme 1 illustrates the relationship between exemplary compounds offormulae I-V. Note that Scheme 1 depicts the relationships between theS-enantiomers. The same relationships exist between the correspondingR-enantiomers. A wide variety of natriuretic compounds within the scopeof the instant invention can be obtained in the manner illustrated.

Formulations of medicaments comprising γ-tocopherol and LLU-α,(S)-LLU-α, or other γ-tocopherol derivatives, detailed above, are asfollows. Racemic LLU-α is synthesized or isolated and may be present asthe free acid or a simple ester. Racemic LLU-α is added to the differingconcentrations of γ-tocopherol with or without a suitable filler. Asupplement comprising γ-tocopherol and racemic LLU-α preferably contains5% to 95% (weight to weight) γ-tocopherol mixed with 5% to 95% racemicLLU-α, and may also include other tocopherols. More preferably, thecompositions of this embodiment of the invention include between 25% and60% racemic LLU-α, or still more preferably no more than 50% (weight toweight) racemic LLU-α. A particularly preferred composition includes 26%(weight to weight) racemic LLU-α with the remaining amount of thesupplement being composed of tocopherols and a suitable filler, with atleast 65% of the tocopherols being γ-tocopherol.

Soft gelatin capsules comprising commercially available γ-tocopherol arefortified with (S)-LLU-α using the same compositions, above. (S)-LLU-αis synthesized or isolated, as detailed above or in the followingexamples, and may be present as the free acid or a simple ester.(S)-LLU-α is added to the formulations of the γ-tocopherol supplementsmentioned above with or without a suitable filler. A supplementcomprising γ-tocopherol and (S)-LLU-α preferably contains 5% to 95%(weight to weight) γ-tocopherol mixed with 5% to 95% (S)-LLU-α, and mayalso include other tocopherols. More preferably, the compositions ofthis embodiment of the invention include between 25% and 60% (S)-LLU-α,or still more preferably no more than 50% (weight to weight) (S)-LLU-α.A particularly preferred composition includes 26% (weight to weight)(S)-LLU-α with the remaining amount of the supplement being composed oftocopherols and a suitable filler, with at least 65% of the tocopherolsbeing γ-tocopherol.

Alternatively, soft gelatin capsules comprising commercially availableγ-tocopherol are fortified with a γ-tocopherol derivative. Theγ-tocopherol derivative is synthesized or isolated, as detailed above orin the following examples, and may be present as the free acid or asimple ester. An γ-tocopherol derivative is added to the formulations ofthe γ-tocopherol supplements mentioned above with or without a suitablefiller. A supplement comprising γ-tocopherol and a γ-tocopherolderivative preferably contains 5% to 95% (weight to weight) γ-tocopherolmixed with 5% to 95% γ-tocopherol derivative, and may also include othertocopherols. More preferably, the compositions of this embodiment of theinvention include between 25% and 60% γ-tocopherol derivative, or stillmore preferably no more than 50% (weight to weight) γ-tocopherolderivative. A particularly preferred composition includes 26% (weight toweight) γ-tocopherol derivative with the remaining amount of thesupplement being composed of tocopherols and a suitable filler, with atleast 65% of the tocopherols being γ-tocopherol. Other tocopherols canbe included in the formulations, including (α-tocopherol, β-tocopheroland δ-tocopherol. In certain circumstances, δ-tocopherol can substitutefor γ-tocopherol in the formulations and methods described herein.

The preferred method of administering principally γ-tocopherol or theformulation comprising γ-tocopherol and racemic LLU-α, (S)-LLU-α, orγ-tocopherol derivative is orally via soft gelatin capsules, however,several methods of administering these therapeutics would be within theskill of one in the art. Gamma-tocopherol or the formulations mentionedabove can be administered neat, as mixtures with other physiologicallyacceptable active or inactive materials such as moistening agents,flavoring agents, binding agents, and extenders, as well as othercompounds having pharmacological activities, such as other diureticswhich increase the distal delivery of sodium, other anti-cancertherapeutics, other high blood pressure medicaments, otheranti-hypertensive agents, or other mixtures of tocopherols. It may alsobe administered with physiologically suitable carriers such as, forexample, olive oil, sesame oil, or other lipid. The compounds can beadministered orally or parenterally, for example, by injection.Injection can be subcutaneous, intravenous, or by intramuscularinjection.

The total daily dose of 200-800 mg can consist of a single individualdose or multiple doses given at intervals. Dosages within these rangescan also be administered by constant infusion over an extended period oftime, usually exceeding 24 hours, until the desired therapeutic benefitshave been obtained. Amounts of the compounds described herein which aretherapeutically effective against specific diseases can also bedetermined through routine investigation.

The following examples are intended to illustrate, but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may be alternativelyemployed. In the examples, the following abbreviations are used:

EI electron impact

FR furosemide response

FT-IR Fourier-transform infrared spectroscopy

HPLC high performance liquid chromatography

MAP mean arterial pressure

MDBK Madin-Darby bovine kidney

MS mass spectrometry

NMR nuclear magnetic resonance

PBS phosphate buffered saline

R_(n) natriuretic ratio

RP-HPLC reverse-phase high performance liquid chromatography

SR sample response

UNaV urine concentration of sodium X urine volume per time

ISOLATION OF NATRIURETIC COMPOUND EXAMPLE 1

Human uremic urine was initially processed by ultrafiltration (3 kDa)and lyophilization, followed by isolation of the post-salt fraction fromSephadex G-25 gel filtration chromatography, following the procedure ofBenaksas et al., Life Sci. 52, 1045-1054 (1993), the entire disclosureof which is herein incorporated by reference. See Table I (firstpurification step).

The crude material was further purified by one of two procedures. Oneprocedure involved four sequential HPLC steps, and the second procedureincluded organic solvent extraction followed by up to five sequentialHPLC steps. Table I summarizes the two methods.

TABLE 1 Summary of steps used in the chromatographic and extractionisolation procedures Puri- fication Chromatographic Extraction StepMethod Method First 3K ultrafiltration, 3K ultrafiltration,lyophilization lyophilization and G-25 and G-25 Second 0.2 M pyridiniumacetate pH Sequential extraction with 5.5/Methanol C₁₈ RP-HPLCisopropanol/diethyl ether yielding soluble compounds Third 1st 0.2 Macetic acid/ 1st 0.2 M acetic acid/methanol methanol C₁₈ RP-HPLC C₁₈RP-HPLC Fourth 2nd (modified) 0.2 M acetic 2nd (modified) 0.2 M aceticacid/methanol C₁₈ RP-HPLC acid/methanol C₁₈ RP-HPLC^(b) FifthIsopropanol/hexane^(a) Isocratic 0.2 M acetic acid/methanol^(c) SixthIsopropanol/hexane silica gel HPLC Seventh 50 mM aceticacid/acetonitrile C₁₈RP-HPLC^(d) ^(a)Amount of resulting material ofLLU-γ was so small that further purification was not pursued. ^(b)LLU-γwas further purified by a chromatography step not used in the mainpurification scheme. ^(c)This HPLC step was only used for isolation ofLLU-α. ^(d)LLU-α methyl ester was also purified using these HPLCconditions.

1. Chromatographic Isolation Method

A four-step sequential HPLC procedure was employed which was amodification of the procedure reported by Benaksas et al., noted above.The first C₁₈ RP-HPLC (Table 1, step 2) was performed on a BeckmanUltrasphere ODS column (10 μm; 21.2×150 mm) eluting at 6 mL/minute witha gradient of 0.2 M pyridinium acetate, pH 5.5 (A) and methanol (B) (80%A:20% B for 22 minutes, a linear gradient to 40% A:60% B over 48minutes, a linear gradient to 100% B over 10 minutes). The column waswashed with 70% toluene:30% methanol, then re-equilibrated at initialconditions for at least 20 minutes. This column wash method wasimplemented in every chromatography employing a methanol eluant. Theeluant was monitored with a Beckman 166 UV detector at 290 nm. Eighty(80) one-minute fractions were collected and dried under reducedpressure in a centrifugal vacuum concentrator.

Based on bioassay evaluation (see Example 2, below) and chromatographiccomparison of previous HPLC runs, fractions 50-80, were combined for thesecond RP-HPLC step (Table 1, third step). A Beckman Ultrasphere ODS(C₁₈) column (5 μm; 10×250 mm) was eluted at 2 mL/minute with a gradientof 0.2 M acetic acid (A), methanol (B) and 70% toluene: 30% methanol(C), (60% A:40% B for 5 minutes, a linear gradient to 50% A:50% B over 5minutes, a linear gradient to 30% A:70% B over 55 minutes, a lineargradient to 100% B over 2 minutes, 100% B for 3 minutes, 100% C for 8minutes, 100% B for 7 minutes). The eluant was monitored forfluorescence (exc. 310-410 nm; emm. 475-610 nm: Beckman 157 detector)and absorbance at 290 nm with a Beckman 168 diode array detector.Ultraviolet spectra were collected by diode array at 2 second intervalsover the range of 202-390 nm. Eighty (80) one-minute fractions werecollected.

As discussed in detail in Example 2, two natriuretically active isolates(LLU-α and LLU-γ) in particular were identified. The region encompassingthe two natriuretically active isolates was pooled and rechromatographedusing a modified acetic acid/methanol gradient for the third RP-HPLC(Table 1, fourth step). The solvents and column were the same as thesecond RP-HPLC above; however, the gradient was changed (60% A:40% B for5 minutes, a linear gradient to 40% A:60% B over 5 minutes, a lineargradient to 30% A:70% B over 28 minutes, a linear gradient to 100% Bover 2 minutes, 100% B for 3 minutes, 100% C for 8 minutes) and onlyfifty (50) one-minute fractions were collected.

During the first aqueous acetic acid-methanol RP-HPLC step (Table 1,third step), chromophore markers corresponding to natriuretically activematerials could be identified when processing different batches ofurine. By rechromatographing fractions 38-58 and 63-66 using a modifiedacetic acid-methanol method (Table 1, fourth step) employing a shortergradient, the two natriuretically active marker chromophores, designatedLLU-α and LLU-γ, reproducibly eluted at 27.8 and 35.4 minutes,respectively. This fourth purification step allowed consistentidentification of natriuretically active crude isolates.

The LLU-α natriuretic isolate was subjected individually to normal phasechromatography on silica gel (Beckman Ultrasphere, 5 μm, 10×250 mm)eluting at 2 mL/minute with a hexane (B) isopropanol (A) gradient (6%A:94% B for 25 minutes, a linear gradient to 100% A over 30 minutes,100% A for 20 minutes, a linear gradient to 6% A:94% B over 5 minutesand an equilibration period at 6% A:94% B for 35 minutes). Seventy (70)one-minute fractions were collected from this fifth purification step(Table 1). Fluorescence was monitored as described above. The wavelengthmonitored for each of the isolates was selected based upon itsabsorbance spectrum from the prior chromatogram. Chromatography of thefirst Isolate (LLU-α) was monitored at 295 nm and that of the second(LLU-γ) at 267 nm. Fractions exhibiting UV absorbance characteristic ofLLU-α and LLU-γ were bioassayed (see below).

2. Extraction Method

Freeze-dried material obtained from the gel filtration chromatographywas stirred with 9 volumes of isopropanol for 18 hours. The isopropanolsolution was then removed and evaporated to dryness on a rotaryevaporator under reduced pressure. The resulting thick, dark brown oilfrom the isopropanol soluble phase was weighed and then alternatelystirred and sonicated for 6 hours and finally stirred for an additional18 hours, with 10 volumes of diethyl ether. The ether solution was thendecanted and 4 volumes of ether were added to the remaining insolublematerial. After stirring for 72 hours, the ether solution was againdecanted. Two volumes of deionized distilled water and 2 volumes ofdiethyl ether were added to the residue. After stirring for 2 hours, theether phase was separated and the aqueous phase was washed three timeswith one volume of ether. The combined ether extracts were washed withsaturated aqueous NaCl and water, and taken to dryness on a rotaryevaporator under reduced pressure. The residue was redissolved in 95%ethanol and again taken to dryness.

The ether extraction residue was dissolved in 40% aqueous methanol andsubjected to acetic acid-methanol RP-HPLC (Table 1, third step). Thechromatographic region from LLU-α to LLU-γ, as identified by theircharacteristic UV spectra, was pooled, dried, re-suspended andchromatographed on the second modified acetic acid-methanol RP-HPLC(Table 1, fourth step). Only LLU-α and LLU-γ were detected after thischromatography step.

Isocratic acetic acid-methanol RP-HPLC (Table 1, fifth step) was thenperformed on LLU-α. Employing a Beckman Ultrasphere ODS (C₁₈) column (5μm; 10×250 mm), LLU-α was eluted at 2 mL/minute with 45% 0.2 M aceticacid and 55% methanol for 35 minutes collecting seventy (70) half-minutefractions. The eluant was monitored for absorbance at 290 nm (diodearray) and fluorescence. LLU-α was identified by its UV spectrum andsubjected to silica gel HPLC (Table 1, sixth step).

The fractions containing LLU-α from the silica gel HPLC were pooled andsubjected to another C₁₈ RP-HPLC step. In this seventh purification step(Table 1), a Beckman Ultrasphere ODS column (5 μm; 4.6×250 mm) waseluted at 1 mL/minute with a gradient of 50 mM acetic acid (A) and 45 mMacetic acid in acetonitrile (B) (85% A:15% B for 3 minutes, a lineargradient to 100% B over 42 minutes, 100% B for 5 minutes). The columnwas washed with 1:1 methylene chloride: acetonitrile for 5 minutesfollowed by re-equilibration at initial conditions for 16 minutes.Chromatography was monitored at 265 and 295 nm with the diode arraydetector. Fifty (50) half-minute fractions were collected staring at 10minutes.

The extraction purification procedure increased the yield of isolatedLLU-α by about 50%. In the chromatographic procedure, encompassing atotal of five purification steps, less than 1 mg of LLU-α was obtainedfrom about 105 g of lyophilized G-25 material (yield less than 9×10⁻⁴%).Approximately 1.8 mg of LLU-α resulted from the extraction procedure(seven purification steps) applied to about 155 g of lyophilized G-25product (yield approximately 1.2×10⁻³%). The two additional RP-HPLCsteps of this procedure led to essentially pure LLU-α. Likewise, theyield of LLU-γ appeared to increase comparably.

LLU-γ from the modified acetic acid-methanol RP-HPLC chromatography step(Table 1, fourth step) can be further purified using a method compatiblefor LC-MS. In this purification step, a Beckman Ultrasphere ODS column(5 μm, 4.6×250 mm) was eluted isocratically at 1 mL/minute with 0.1%trifluoroacetic acid, 40% acetonitrile, and 60% water for 30 minutes.LLU-γ from the previous chromatographic step elutes at 16.5 minutes.Between runs the column is washed with 0.1% trifluoroacetic acid inacetonitrile for 10 minutes, followed by reequilibration at initialconditions for 10 minutes. Chromatography was monitored at 265 and 230nm with a diode array detector. LLU-γ was collected as a singlefraction.

TABLE II Chemical characteristics of the natriuretic LLUs LLU-α LLU-γExact Mass 264.1373 ND^(a) Empirical C₁₅H₂₀O₄ ND Formula UV λmax 205 nmλmax 220 nm Characteristics λmax 294 nm λmax 268 nm Functional carboxylND Groups Determined hydroxyl by IR aryl ether Physical Unstable inUnstable when Properties dilute Base Purified Unstable in Very Unstablein CDCl₃ Dilute Base Reaction with HNF-α methyl ester ND CH₂N₂C₁₄H₁₉O₂CO₂CH₃ MW 278.1515 + Other Products ^(a)ND: Not Determined

Isolated from early fractions of silica gel HPLC of LLU-α was the drugnaproxen, which was being administered to some urine donors. Itsidentity was determined by NMR and verified by comparison with the NMRspectrum of commercial naproxen. Naproxen serves as an additional markerduring the silica gel HPLC.

3. Treatment of LLU-α with CH₂N₂

Diazomethane was generated by treatment of1-methyl-3-nitro-1-nitrosoguanidine (112 mg, 760 μmol) with 400 μL 50%KOH (aq). The diazomethane was distilled into 1 mL diethyl ether at −7°C. This solution was then added to 700 μg (2.6 μmol) LLU-α in 0.5 mLdiethyl ether at 0° C. The reaction mixture was warmed to ambienttemperature, then allowed to stand for 40 minutes. Solvent was removedunder a stream of N₂ and the residue dissolved in 15% 45 mM acetic acidin acetonitrile/85% 50 mM acetic acid and subjected to the acetic acid-acetonitrile RP-HPLC purification step as described above (seventhstep). The approximate yield of the ester was 53%. Methyl esterificationof LLU-α followed by RP-HPLC yielded essentially pure LLU-α methylester. The methyl ester was synthesized to further the characterizationof LLU-α. LLU-α methyl ester eluted as an apparently homogenous singlepeak from acetic acid-acetonitrile RP-HPLC. A total of approximately 0.9mg of LLU-α methyl ester was isolated and subjected to chemicalcharacterization by ultraviolet, infrared, ¹³C- and ¹H-NMR and massspectroscopy. The physical chemical characteristics, molecular weightand inferred molecular formula of both LLU-α and its methyl ester arelisted in Table II.

BIOASSAYS FOR BIOLOGICAL ACTIVITY EXAMPLE 2

1. In Vivo bioassay

The assay for natriuresis in conscious rats has been describedpreviously (see Benaksas et al., above). The assay is briefly reiteratedhere. Female Sprague-Dawley (Harlan) rats (200-250 g) were cannulated inthe femoral artery and vein for monitoring of mean arterial pressure(MAP) and Infusion of saline and samples, respectively. The bladder wascatheterized for collection of urine in ten-minute periods. Furosemide(0.4 mg/kg bwt; 1 mg/mL in 0.17% saline) was infused as a positivecontrol at the beginning of the sixth ten-minute period. The sample wasinfused at the beginning of the seventeenth ten-minute period. Urine wascollected for another 150 minutes. The volume of the urine wasdetermined gravimetrically and the Na⁺ and K⁺ concentrations determinedwith a Beckman E2A electrolyte analyzer. From these data the sodiumexcretion values (UNaV) were calculated.

The natriuretic response of a sample was normalized to the dose offurosemide infused. The not sodium excretion for the Infusion offurosemide or sample was calculated as follows. The median sodiumexcretion value (μmoles Na⁺/10 minute period) for the five periodsbefore infusion of furosemide or sample was used to establish a baselinevalue for the calculation of ΔUNaV (=μmoles Na⁺ period−baseline μmolesNa⁺) for administration of either furosemide or sample respectively. Thesum of ΔUNaV for the four periods following infusion of furosemide wasthe net sodium excreted for furosemide, defined as FR The sum of ΔUNaVfor the fifteen periods following infusion of the sample was the netsodium excreted for the sample defined as SR. The natriuretic ratioR_(n) (or normalized natriuretic response) of a sample was calculated bydividing SR by FR (R_(n)=SR/FR). A sample is considered natriureticallyactive if the R_(n) value for that sample was greater than or equal to0.67 (greater than 99% confidence limits).

Partially purified LLU-α from silica gel-HPLC (sixth purification step)was assayed for natriuretic activity utilizing the in vivo bioassay. Itwas active in the 4-8 μg/kg dose range and showed no activity at loweror higher doses (Table III). LLU-α is also at 8 μg/kg when evaluated inthe in vivo bioassay after being further purified on aceticacid/acetonitrile RP-HPLC (seventh step of extraction method).

TABLE III Dose response of LLU-α present in fractions from the silicagel HPLC step of the extraction procedure from uremic urine FractionDose (μg) Natriuretic Response (R)^(a) 17 0.2 −0.14 1 0.27 2 1.14 2 0.7510 0.26 18 56.4 0.23 22.4 0.02 19 0.2 0.24 1 0.93 2 −0.10 2 0.82 10 0.0920 2 1.32 2 0.39 21 2 −0.06 2 0.39 ^(a)Natriuretic ratio greater than0.67 indicates that a sample is natriuretically active (99% confidencelimits).

LLU-α and -γ when infused into the rat produced sustained natriuresiswith no effect on blood pressure. LLU-γ has not been purifiedsufficiently to obtain a dose-response curve for natriuresis, owing toits instability. LLU-α displays a narrow and biphasic natriureticdose-response curve (Table III). There was no detectable kaliuresis whenLLU-α was infused. Some kaliuresis occurred after the infusion of LLU-γ,however, this was not always observed. Neither LLU-α nor -γ caused asignificant Change in mean arterial pressure.

2. Na⁺/K⁺-ATPase Inhibition assay

The assay in MDBK cells has been described previously (see Benaksas atal., above). The assay is described briefly here. Madin-Darby bovinekidney (MDBK) cells (ATCC:CCL22) were maintained in Dulbecco's ModifiedEagle's Medium (DMEM) with 5% Fetal Bovine Serum and 5% Bovine CalfSerum in a 5% CO₂/95% humidified air atmosphere at 37° C. and split(1:2) once per week.

One day before the assay, cells were plated in a 96-well plate at adensity of 5×10⁵ cells/well in DMEM with serum. On the day of the assaythe medium was removed and the cells washed with phosphate bufferedsaline (PBS) before addition of 100 μL of assay media (122 mM NaCl, 1.8mM CaCl₂, 0.8 mM MgSO₄, 24 mM NaHCO₃, 1 mM sodium pyruvate, 25 mMglucose, 14 mM glycylglycine, 0.2% phenol red, 8 mM Na₂HPO₄ 1.15 mMKH₂PO₄, pH 8.0) and 100 μl of sample. The plate was preincubated for 30minutes at 37° C., then chilled on ice for 10 minutes. To each well wasadded 0.15 μCi ⁸⁶RbCl (Amersham) in 10 μL of assay media. The plate wasthen incubated at 37° C. for 10 minutes. A portion (100 μL) of thesupernatant was counted with 0.5 mL of scintillation cocktail in aliquid scintillation counter. As a control for Na⁺/K⁺-ATPase inhibition,a dose response curve for ouabain in the range of 10⁻⁵-10⁻⁸ M wasobtained. Intra-experiment coefficient of variation for ouabain was3-15%. Inhibition of ⁸⁶Rb⁺ uptake by samples was corrected for thatuptake which was inhibitable by ouabain.

When LLU-α was assayed in the Na⁺/K⁺-ATPase inhibition assay itexhibited no inhibition in the range of 0.2-200 ng/well. Assay of crudeLLU-γ obtained from the acetic acid-methanol RP-HPLC rechromatographystep in the sodium pump inhibition assay showed no inhibition of thesodium pump.

ANALYTICAL SPECTROSCOPY EXAMPLE 3

In addition, spectroscopy other than UV was performed. ¹³C- and ¹H-NMRspectra were recorded at 500.1357 MHz in deutero-Chloroform (99.9%) in aGN-500 spectrometer (General Electric). High resolution Electron-Impact(ED mass spectra with a resolution of 2000 were recorded at anionization voltage of 70 eV, source temperature of 220° C. andintroduction of sample by direct probe on a VG7070 EHF high resolutionmass spectrometer. Fourier-transform infrared (FT-IR) spectroscopy wasperformed on a Nicolet 5DX with 4 wavenumber resolution.

The IR and ¹³C-NMR spectra of LLU-α provided evidence for the presenceof a carboxylic acid group. This explained the tailing of LLU-α observedupon elution from isopropanol/hexane silica gel HPLC (sixth purificationstep). The presence of a carboxyl group was verified when the reactionof LLU-α with diazomethane resulted in a product that was less polar onRP-HPLC and had an exact mass 14 units greater than LLU-α as determinedby MS (Table II). This is consistent with the formation of a methylester.

In the following synthesis examples, Examples 4-10 set forth generalmethods useful to produce a wide range of compounds within the scope ofthe invention. Examples 11-21 describe syntheses of specific compounds.

SYNTHESIS OF RACEMIC 6-HYDROXYCHROMANS EXAMPLE 4

To a solution of hydroquinone 1 (0.01 mol) and a catalyst, preferablyboron trifluoride diethyl etherate (0.016 mol) In an organic solvent,preferably dry dioxane (10 mL), is added vinyl lactone 2 (0.016 mol) inan organic solvent, preferably dry dioxane (5.0 mL) over 1-60 minutes,preferably 60 minutes, at 0-150° C., preferably 110° C., under an inertgas. The reaction mixture is stirred for 0 to 8 hours, preferably 0hours, at the selected temperature, cooled to room temperature, anddiluted with an organic solvent, preferably diethyl ether (200 mL) Thereaction mixture is then washed with water (100 mL, 2×50 mL), dried oversodium sulfate (Na₂SO₄), and solvent is removed under reduced pressureto afford a brown oily residue. The residue is dissolved in alcohol,preferably methanol (30 mL), and the alcohol is then removed underreduced pressure. The brown oily liquid or semisolid is further purifiedby chromatography, preferably on silica gel, to afford pure racemicchroman derivative 3.

SYNTHESIS OF RACEMIC CHROMANS EXAMPLE 5

To a solution of phenol 4 (0.01 mol) and a catalyst, preferably borontrifluoride diethyl etherate (0.016 mol) in an organic solvent,preferably dry dioxane (10 mL), Is added vinyl lactone 2 (0.016 mol) Inan organic solvent, preferably dry dioxane (5.0 mL) over 1-60 minutes,preferably 60 minutes, at 0-150° C., preferably 110° C., under an inertgas. The reaction mixture is stirred for 0 to 8 hours, preferably 0hours, at the selected temperature, cooled to room temperature, anddiluted with an organic solvent, preferably diethyl ether (200 mL). Thereaction mixture is then washed with water (100 mL, 2×50 mL), dried oversodium sulfate (Na₂SO₄), and solvent is removed under reduced pressureto afford a brown oily residue. The residue is dissolved in alcohol,preferably methanol (30 mL), and the alcohol is then removed underreduced pressure. The brown oily liquid or semisolid is further purifiedby chromatography, preferably on silica gel, to afford pure racemicchroman derivative 5.

SYNTHESIS OF CHROMAN METHYL ESTERS EXAMPLE 6

Chroman 3 (R₃═OH) or 5 (see Examples 4 and 5 above) (0.01 mol) isdissolved in methanol (30 mL), and a solution of diazomethane in etheris added at 0-5° C. until the yellow color of the diazomethane remains.The reaction mixture is left at room temperature for 2-5 hours, solventis removed, and the desired product 6 is crystallized from a suitableorganic solvent.

SYNTHESIS OF CHROMAN ESTERS EXAMPLE 7

Chroman 3 (R₃═OH) or 5 (10 mmol) is dissolved in dry tetrahydrofuran (30mL) with an alcohol R₇—OH (12 mmol), 1-hydroxybenzotriazole (10 mmol)and 1,3-dicyclohexylcarbodilmide (11 mmol) at 2-5° C. The reactionmixture is stirred at 2-5° C. for one hour and at 23° C. for one to 20hours. Precipitated dicyclohexyl urea is filtered, solvent is removedunder reduced pressure, and the residue is diluted with ethyl acetate(150 mL). The organic phase is washed with aqueous KHSO₄ (10%, 40 mL),water (50 mL) and saturated aqueous hydrogen carbonate (50 mL), and thendried over sodium sulfate. The solvent is removed under reducedpressure, and the residue is purified by chromatography, preferablysilica gel, to afford pure racemic ester 7.

SYNTHESIS OF CHROMAN AMIDES EXAMPLE 8

Chroman 3 (R₃═OH) or 5 (10 mmol) is dissolved in dry tetrahydrofuran (40mL), and neutralized with N-methylmorpholine. isobutyl chlorocarbonate(10 mmol) is added, followed one minute later by a selected amine(R₇—NH₂ or R₇R₈—NH), or ammonia (11 mmol). The reaction mixture isallowed to reach room temperature. After stirring at room temperaturefor 1 hour, THF is removed under reduced pressure, and the residue istaken into ethyl acetate (250 mL). The ethyl acetate solution issuccessively washed with aqueous KHSO₄ (10%, 40 mL), water (50 mL), andsaturated aqueous hydrogen carbonate (50 mL), and then dried over sodiumsulfate. The solvent is removed under reduced pressure, and the residueIs purified by chromatography, preferably silica gel, to afford pureracemic amide 8.

SYNTHESIS OF R₄ CHROMAN ESTERS EXAMPLE 9

Method 1:

Chroman 3 (R₃═OH) or 5 (10 mmol) is dissolved in pyridine (20 mL). andacid anhydride (30 mmol) is added at 5° C. The reaction mixture is leftat room temperature for 18 hours, solvent is removed in vacuum, and theresidue is dissolved in ethyl acetate (100 mL). washed with citric acid(10%, 30 mL). and water (30 mL). and dried over sodium sulfate. Thesolvent is removed and the residue is crystallized from ethylacetate/hexane to afford ester 9.

Method 2:

Chroman 3 (R₃═OH) or 5 (10 mmol) is dissolved in dry pyridine (50 mL)under nitrogen and cooled in an ice-water bath. Acyl chloride (10 mmol)is added via syringe over 15 minutes. Stirring is continued for 1 hourat room temperature. Pyridine is removed under reduced pressure, and theresidue is dissolved in ethyl acetate (100 mL). The ethyl acetate phaseis washed with water (2×40 mL). aqueous hydrochloric acid (0.05 M, 30mL) and water (40 mL). and dried over sodium sulfate. The solvent isremoved under reduced pressure, and the residue is purified bychromatography, preferably on silica gel, to afford ester 9.

SYNTHESIS OF OXIDIZED CHROMAN DERIVATIVES EXAMPLE 10

Chroman 3 or 5 (0.3 mmol) is dissolved in methanol (2.5 mL) in a flask.A ferric chloride solution is prepared by dissolving 1.0 g FeCl₃-6H₂O inwater (4.0 mL) and adding methanol (4.0 mL) The ferric chloride solution(2.5 mL) Is added to the flask at room temperature with vigorousstirring for 30 minutes in darkness. Methanol 13 removed in vacuum, andthe residue Is dissolved In ether (70 mL) The ether solution is washedwith water (3×20 mL) and dried over sodium sulfate, then the solvent isremoved. The product is purified on an RP HPLC column (CH₃CH/H₂Ogradient) to afford a yellow-to-brown oily product.

SYNTHESIS OF RACEMIC 2,7,8-TRIMETHYL-2-(β-CARBOXYETHYL)-6-HYDROXYCHROMAN (LLU-α) EXAMPLE 11

To a solution of 2,3-dimethyl-1,4-hydroquinone (0.01 mol) and borontrifluoride diethyl etherate (0.016 mol) in dioxane (10 mL, dried onsodium) in a flask was added γ-methyl-γ-vinylbutyrolactone (0.016 mol)in dioxane (5.0 mL) over 50 min at 110° C. (oil bath, reflux) undernitrogen. The reaction mixture was cooled to room temperature anddiluted with ether (200 mL), then washed with water (100 mL, 2×50 mL)and dried over sodium sulfate. Ether was then removed under reducedpressure to afford a brown, oily residue. The residue was dissolved inmethanol (30 mL) and solvent was removed under reduced pressure. Theresidue was redissolved in methanol (10 mL) and the flask was purgedwith nitrogen and stored at 5° C. for 20 hours. The resulting suspensionwas centrifuged, and the supernatant was removed. The remaining whitesolid (see Example 21, below) was suspended in aqueous 70% methanol (15mL) and again centrifuged. The supernatant was combined with theprevious supernatant, and methanol was removed in vacuum to afford abrown, oily liquid. The liquid was further purified by flash columnchromatography on silica gel (eluent ethyl acetate/hexane/acetic acid,500:300:1) to afford pure racemic2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxy chroman, which wascrystallized from ether-hexane in a yield of 40%. M.P.: 147-148° C.

SYNTHESIS OF RACEMIC 2,5,7,8-TETRAMETHYL-2-(β-CARBOXYETHYL)-6-HYDROXYCHROMAN EXAMPLE 12

To a solution of 2,3,5-trimethyl-1,4-hydroquinone (0.01 mol) and borontrifluoride diethyl etherate (0.016 mol) in dioxane (10 mL, dried onsodium) in a flask was added γ-methyl-γ-vinylbutyrolactone (0.016 mol)in dioxane (5.0 mL) over 50 min at 110° C. (oil bath, reflux) undernitrogen. The reaction mixture was cooled to room temperature anddiluted with ether (200 mL), then washed with water (100 mL, 2×50 mL)and dried over sodium sulfate. Ether was then removed in vacuum. Theresidue was dissolved in methanol (30 mL), and solvent was removed invacuum. The brown, oily residue was dissolved in methanol (20 mL), andwater was added until the solution became turbid (app. 20 mL), then theflask was purged with nitrogen and stored overnight in a refrigerator.The light yellow solid was filtered on a sinter funnel, washed withaqueous 50% methanol and dried in a dessicator. The product was furtherpurified by flash column chromatography on silica gel (eluent ethylacetate/hexane/acetic acid, 500:300:1) to afford pure racemic2,5,7,8-tetramethyl-2-(β-carboxyethyl)-6-hydroxy chroman, which wascrystallized from ether-hexane in a yield of 50%. M.P.: 173° C.

SYNTHESIS OF RACEMIC 2,5,7,8-TETRAMETHYL-2-(β-CARBOXYETHYL)-CHROMANEXAMPLE 13

To a solution of 2,3,5-trimethylphenol (0.01 mol) and boron trifluoridediethyl etherate (0.016 mol) in dioxane (10 mL, dried on sodium) in aflask was added γ-methyl-γ-vinylbutyrolactone (0.016 mol) in dioxane(5.0 mL) via syringe pump over 50 min at 110° C. (oil bath, reflux)under nitrogen. The reaction mixture was cooled to room temperature anddiluted with ether (200 mL), then washed with water (100 mL, 2×50 mL)and dried over sodium sulfate. Ether was then removed in vacuum. Theresidue was dissolved in methanol (30 mL) and solvent was removed invacuum. The reaction mixture was purified by flash column chromatographyon silica gel (eluent ethyl acetate/hexane, 1:1). Fractions containingthe desired chroman were pooled, solvent was removed, and the compoundwas crystallized from ethyl acetate/hexane to afford a white crystallineproduct in a yield of 40%. M.P.: 148-149° C.

SYNTHESIS OF RACEMIC 2,7,8-TRIMETHYL-2-(β-CARBOXYETHYL)-CHROMAN EXAMPLE14

To a solution of 2,3-dimethylphenol (0.01 mol) and boron trifluoridediethyl etherate (0.016 mol) in dioxane (10 mL, dried on sodium) in aflask was added γ-methyl-γ-vinylbutyrolactone (0.016 mol) in dioxane(5.0 mL) via syringe pump over 50 min at 110° C. (oil bath, reflux)under nitrogen. The reaction mixture was cooled to room temperature anddiluted with ether (200 mL) then washed with water (100 mL, 2×50 mL) anddried over sodium sulfate. Ether was then removed in vacuum. The residuewas dissolved in methanol (30 mL), and solvent was removed in vacuum.The reaction mixture was purified by flash column chromatography onsilica gel (eluent ethyl acetate/hexane, 1:1). Fractions containing thedesired chroman were pooled, solvent was removed, and the compound wascrystallized from ethyl acetate/hexane. M.P.: 93-94° C.

SYNTHESIS OF RACEMIC 4-METHYL-6-(5,6-DIMETHYLBENZOHINOYL)-4-HEXANOLIDEXAMPLE 15

Racemic 2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxychroman (100 mg) wasdissolved in methanol (2.5 mL) in a flask. A solution of ferric chloridewas prepared by dissolving 1.0 g FeCl₃-6H₂O in water (4.0 mL) and addingmethanol (4.0 mL) The ferric chloride solution (2.5 mL) was added to theflask at room temperature with vigorous stirring in darkness for 30minutes. Methanol was removed in vacuum, and the residue was dissolvedIn ether (70 mL). The ether solution was washed with water (3×20 mL)dried over sodium sulfate, and the solvent was removed. The product waspurified on an RP HPLC column (CH₃CN/H₂O gradient) to afford ayellow-to-brown oily product in 60% yield.

SYNTHESIS OF RACEMIC4-METHYL-6-(3,5,6-TRIMETHYLBENZOCHINOYL)-4-HEXANOLID EXAMPLE 16

Racemic 2,5,7,8-tetramethyl-2-(β-carboxyethyl)-6-hydroxychroman (100 mg)was dissolved in methanol (2.5 mL) in a flask. The ferric chloridesolution of Example 10 (2.5 mL) was added to the flask at roomtemperature with vigorous stirring in darkness for 30 minutes. Methanolwas removed in vacuum, and the residue was dissolved in ether (70 mL)The ether solution was washed with water (3×20 mL) dried over sodiumsulfate, and the solvent was removed. The product was purified on an RPHPLC column (CH₃CN/H₂O gradient) to afford a yellow-to-brown oilyproduct in 60% yield.

RESOLUTION OF RACEMIC 2,7,8-TRIMETHYL-2-(β-CARBOXYETHYL)-6-HYDROXYCHROMAN (LLU-α) EXAMPLE 17

The resolution of (S) and (R)-enantiomers was carried out on an(S,S)-WHELK-O 1 column (Regis Technologies, Inc.) 250×4.6 mm, 1 mL/min,using as eluent isocratic 80% hexane:20% propanol:0.5% acetic acid.Fractions were monitored by UV spectroscopy, collected and dried underan argon stream. The enantiomers elute at 6.8 minutes and 8.7 minutes.Isolated LLU-α, when run on this system, elutes at 8.6 minutes.

SYNTHESIS OF (R)- AND(S)-4-METHYL-6-(5,6-DIMETHYLBENZOCHINOYL)-4-HEXANOLID EXAMPLE 18

(R)-2,7,8-Trimethyl-2-(β-carboxyethyl)-6-hydroxy chroman 0 00 mg) (seeExample 17) was dissolved in methanol (2.5 mL) and ferric chloridesolution (2.5 mL) was added at room temperature with vigorous stirringfor 30 minutes in darkness. Methanol was removed under reduced pressure,and the residue was dissolved in ether (70 mL). The ether solution waswashed with water (3×20 mL) dried over sodium sulfate, and the solventwas removed. The product was purified by HPLC, using a Phenomenex column(SPHEREX 10 ODS, 250×21.2 mm) with CH₃CN-H₂O 50:50 for 5 minutes, lineargradient to CH₃CN—H₂O 90:10 in 30 minutes, linear gradient to 100% CH₃CNin 5 minutes, flow rate 6 mL/min. Fractions containing the desiredoxidation product were identified by UV spectroscopy. The fractions werepooled, and solvent was removed under reduced pressure to afford(R)-4-Methyl-6-(5,6-dimethylbenzochinoyl)-4-hexanolid as a yellow tobrown oil.

The foregoing process was repeated using(S)-2,7,8-Trimethyl-2-(β-carboxyethyl)-6-hydroxy chroman (100 mg) toafford (S)-4-Methyl-6-(5,6-dimethylbenzochinoyl)-4-hexanolid as a yellowto brown oil.

SYNTHESIS OF RACEMIC 2,7,8-TRIMETHYL-2-(β-CARBOXYETHYL)-6-ACETYL CHROMANEXAMPLE 19

Racemic 2,7,8-Trimethyl-2-(β-carboxyethyl)-6-hydroxy chroman (500 mg)(see Example 11) was dissolved in pyridine (20 mL) at room temperature,and acetic anhydride (10 mL) was added. The solution was maintained atroom temperature for 5 hours, solvent was removed under vacuum, methanol(4×10 mL) was added and then removed under reduced pressure. Theresidual oil was dissolved in ethyl acetate (150 mL) and the organicphase was washed with water (50 mL) aqueous HCl (1 N, 50 mL) and water(50 mL) then dried over sodium sulfate. Solvent was then removed, andthe residual oily material was purified on -a silica gel column withhexane/ethyl acetate (1:1). The desired product crystallized fromacetone/hexane, m.p. 105-107° C.

SYNTHESIS OF RACEMIC 2,7,8-TRIMETHYL-2-(β-CARBOXYETHYL)-6-ACETYL CHROMANMETHYL ESTER EXAMPLE 20

Racemic 2,7,8-Trimethyl-2-(β-carboxyethyl)-6-acetyl chroman (500 mg)(see Examples 11 and 19) was dissolved in methanol (10 mL), and etheraldiazomethane was added until the yellow color of diazomethane remained.The solution was maintained at room temperature for 1 hour, solvent wasremoved, and the residue was purified on a silica gel column withhexane/acetone (3:1). The desired product crystallized frommethanol/water, m.p. 87-88° C.

SYNTHESIS OF BENZODIPYRAN METHYL ESTER EXAMPLE 21

Benzodipyran derivative 10 (m.p. 225-227° C.) was isolated as a reactionbyproduct from the synthesis of LLU-α (Example 11). Derivative 10 existsas a racemic mixture of a meso-(R,S) compound and a diastereomeric pair(R,R) and (S,S). Derivative 10 (1.0 g) was suspended in methanol (10mL), and etheral diazomethane was added until the yellow color ofdiazomethane remained. The clear solution was maintained at roomtemperature for 1 hour, solvent was removed, and the residue waspurified on a silica gel column with hexane/acetone (3:1). The desiredproduct crystallized from hexane, m.p. 75-76° C.

TREATMENT AND PREVENTION OF HIGH BLOOD PRESSURE EXAMPLE 22

High blood pressure is a major contributory factor to cardiovascularrelated illness. The administration of a supplement according to thepresent invention, as detailed in the following example, will treat andprevent high blood pressure.

The blood pressure of a patient suffering from high blood pressure isdetermined by conventional methods. The patient is then given a dailydose of supplement (200-400 mg) containing a formulation of γ-tocopherol75% (weight to weight) and LLU-α 25% (weight to weight). The dailycourse of supplementation is continued for a period of 9-12 months afterwhich time the patient's blood pressure is again determined. After aperiod of 12 months, a reduction in blood pressure is observed. As acontrol, placebos or supplements containing equivalent amounts ofα-tocopherol are provided to patients suffering from high bloodpressure. The results of this study will demonstrate thatsupplementation with a formulation of γ-tocopherol and LLU-α will treatand prevent, high blood pressure in a patient suffering from thisdisease to a greater extent than supplementation with a placebo or anequivalent amount of γ-tocopherol.

TREATMENT AND PREVENTION OF THROMBOEMBOLIC DISEASE EXAMPLE 23

Thromboembolic disease is a considerable problem for insulin-dependantdiabetics, the elderly, and people suffering from cardiovasculardisease. The administration of a supplement of the present invention,according to the example below, will treat and prevent thromboembolicdisease.

Blood from a patient suffering from thromboembolic disease is drawn anda platelet aggregation assay, as known by one of skill in the art, isperformed on the sample. (See Richardson and Steiner, Adhesion of HumanPlatelets Inhibited by Vitamin E, Chapter 24, Vitamin E in Health andDisease, Packer and Fuchs editors, Marcel Dekker Inc. Publishers 1993pp. 297-311). The patient is then given a daily dose of supplement200-400 mg containing a formulation of γ-tocopherol 75% (weight toweight) and LLU-α 25% (weight to weight). The daily course ofsupplementation is continued for a period of 2-4 weeks after which timethe patient's blood is again drawn and platelet aggregation isdetermined. After a period of 4 weeks, a reduction in plateletaggregation will be observed. As a control, placebos or supplementscontaining equivalent amounts of α-tocopherol are provided to patientssuffering from thromboembolic disease. The results of this study willdemonstrate that supplementation with a formulation of γ-tocopherol andLLU-α will reduce platelet aggregation and thereby treat and preventthromboembolic disease in a patient suffering from this malady betterthan supplementation with a placebo or an equivalent amount ofα-tocopherol.

REDUCTION OF PLATELET BINDING TO ADHESIVE PROTEINS EXAMPLE 24

Platelet aggregation and thromboembolic disease are related to theaberrant binding of platelets to adhesive proteins. By following theexample disclosed below, platelet binding to adhesive proteins can beinhibited by supplementation of γ-tocopherol and LLU-α.

Blood from a patient suffering from thromboembolic disease is drawn anda platelet adhesion assay, as known by one of skill in the art, isperformed on the sample. (See Richardson and Steiner, Adhesion of HumanPlatelets Inhibited by Vitamin E, Chapter 24 Vitamin E in Health andDisease, Packer and Fuchs editors, Marcel Dekker Inc. Publishers 1993pp. 297-311). The patient is then given a daily dose of supplement100-200 mg containing a formulation of γ-tocopherol 75% (weight toweight) and LLU-α 25% (weight to weight). The daily course ofsupplementation is continued for a period of 2-4 weeks after which timethe patient's blood is again drawn and platelet adhesion is determined.After a period of 4 weeks, a reduction in platelet adhesion will beobserved. As a control, placebos and supplements containing equivalentamounts of α-tocopherol can be provided to patients suffering fromthroboembolic disease. The results of this study will demonstrate thatsupplementation with a formulation of γ-tocopherol and LLU-α will reduceplatelet binding to adhesive protein better than supplementation with aplacebo or an equivalent amount of α-tocopherol.

TREATMENT AND PREVENTION OF ARTHEROSCELEROSIS EXAMPLE 25

Oxidized LDL is chemoattractant to circulating monocytes and inhibitsmacrophage mobility in the intima. Indiscriminate uptake of oxidativelymodified LDL by scavenger receptors of macrophages results incholesterol-laden foam cells and fatty-streak formation. These events,and the potential cytotoxicity of oxidized LDL, further promote theevolution of fatty streaks to a more advanced lesion and cardiovasculardisease. In vitro indices of LDL oxidation are known in the prior artand can be adapted to determine the ability of a formulations ofγ-tocopherol and LLU-α to prevent atheroscelerosis and cardiovasculardisease. The following example provides one approach by which to treatand prevent atheroscelerosis cardiovascular disease.

Blood from a patient suffering from atheroscelerosis is drawn and theamount of oxidized LDL present in the sample is determined. (See Freiand Ames, Relative Importance of Vitamin E in Antiperoxidative Defensesin Human Blood Plasma and Low-density Lipoprotein (LDL), Chapter 10Vitamin E in Health and Disease, Packer and Fuchs editors, Marcel DekkerInc. Publishers 1993 pp. 131-139). The patient is then given a dailydose of supplement 400-800 mg containing a formulation of γ-tocopherol75% (weight to weight) and LLU-α 25% (weight to weight). The dailycourse of supplementation is continued for a period of 2-4 weeks afterwhich time the patient's blood is again drawn and the amount of oxidizedLDL present in the sample is determined. As a control, placebos orsupplements containing equivalent amounts of α-tocopherol are providedto patients suffering from atheroscelerosis. The results of this studywill demonstrate that supplementation with a formulation of γ-tocopheroland LLU-α will reduce the level of oxidized LDL in a patient and therebytreat and prevent artheroscelerosis and cardiovascular disease betterthan supplementation with a placebo or an equivalent amount ofα-tocopherol.

TREATMENT AND PREVENTION OF CANCER EXAMPLE 26

The antioxidant and nitrogen scavenger properties of γ-tocopherol andLLU-α can be used to treat and prevent cancer, as described below. Thefollowing example is based on an experimental methodology accepted bythose of skill in the art to reflect anti-tumor effects in the humanbody. (See Elson, Impact of Palm Oil on Experimental Carcinogenesis,Chapter 39 Vitamin E in Health and Disease, Packer and Fuchs editors,Marcel Dekker Inc. Publishers 1993 pp. 533-545). Four groups of mice areused in the study: (1) control mice in which tumor formation is notinduced but treatment with a formulation of γ-tocopherol 75% (weight toweight) and LLU-α 25% (weight to weight) is rendered; (2) control micein which tumor formation is induced and treatment is not rendered; (3)experimental mice in which tumor formation is induced and treatment withγ-tocopherol 75% (weight to weight) is rendered; and (4) experimentalmice in which tumor formation is induced and treatment with aformulation of γ-tocopherol 75% (weight to weight) and LLU-α 25% (weightto weight) is rendered. As a further control, mice in which tumorformation is induced are treated with varying concentrations ofα-tocopherol so as to evaluate the relative effectivity of γ-tocopheroland the formulation of γ-tocopherol and LLU-α, as compared toα-tocopherol.

Mice which receive treatment with γ-tocopherol or a formulation ofγ-tocopherol and LLU-α, as described above, are given 20 mg/kg ofsupplement for a period of 2-4 weeks. Tumor cells derived from aspontaneously arising mammary tumor are then injected into the thigharea of the experimental mice to induce tumor formation. Treatment withγ-tocopherol and the formulation of γ-tocopherol and LLU-α is continuedaccording to the protocol above. After 21 days, the mean volume oftumors in the mice is determined and compared. The results of this studywill demonstrate that the mean volume of tumors in the mice treated withγ-tocopherol and the formulation of γ-tocopherol and LLU-α, is less thanthe mean volume of tumors in the control mice in which tumor formationwas induced but γ-tocopherol or the formulation of γ-tocopherol andLLU-α is not administered.

REDUCTION IN THE FORMATION OF FREE RADICALS EXAMPLE 27

A reduction in the formation of free radicals is thought to be essentialto prevent cancer and cardiovascular disease. The following exampleprovides an approach to evaluate the efficacy of supplementation with aformulation of γ-tocopherol and LLU-αfor reducing the formation of freeradicals.

Human excretion of breath pentane has been used in a number of humanstudies as a measure of free-radical reactions. (See Packer et al.,Significance of Vitamin E for the Athlete, Chapter 34, Vitamin E inHealth and Disease, Packer and Fuchs editors, Marcel Dekker Inc.Publishers 1993 pp. 465-471). The breath pentane is measured from twogroups of human volunteers. The first group serves as the control forthe study and is not supplemented with a formulation of γ-tocopherol andLLU-α. The second group is supplemented for 2-4 weeks (200-400 mg/day)with a formulation of γ-tocopherol 75% (weight to weight) and 25%(weight to weight) LLU-α. As another control, human volunteerssupplemented with an equivalent amount α-tocopherol can be used. Boththe control and experimental groups are subjected to exhaustive exerciseand a measurement of breath pentane is taken shortly thereafter. Theresults will show that breath pentane, a measure of free-radicalformation in the body, is reduced in humans who received supplementationwith a formulation of γ-tocopherol and LLU-α as compared to a controlgroup which received either no supplementation or supplementation withα-tocopherol.

TREATMENT AND PREVENTION OF NEUROPATHOLOGICAL LESIONS EXAMPLE 28

Vitamin E deficiency in animals is associated with axonal dystrophy thatinvolves degeneration in the posterior cord and in the gracile andcuneate nuclei. Humans who suffer from malabsorption syndromes that areassociated with decreased absorption or transport of vitamin E developsimilar neurological symptoms including hyporflexia, gait disturbances,decreased sensitivity to vibration and proprioception andopthalmoplegia. Neuropathological lesions, including axonal degenerationof the posterior cord and the gracilis nucleus in humans are comparablewith those found in animals deficient in vitamin E. Rats suffering fromvitamin E deficiency can be used to determine the therapeutic benefitsof supplemention with a formulation of γ-tocopherol and LLU-α, accordingto the following example, for the treatment and prevention ofneurological conditions.

Rats are maintained on a vitamin E depleted diet for a period of 8 weeksso that neuropathological lesions are allowed to develop. One group ofvitamin E deficient rats is continued on the vitamin E depleted dietwithout vitamin E supplementation during the course of the study andserves as a control. A second control consists of vitamin E deficientrats maintained on a vitamin E depleted diet but supplemented with 20mg/kg of α-tocopherol. The experimental group of vitamin E deficientrats is treated with either 20 mg/kg of γ-tocopherol or a formulation75% (weight to weight) of γ-tocopherol and 25% (weight to weight) LLU-αfor a period of 2-4 weeks. The rats are then sacrificed and the presenceof neuropathological lesions is determined by methods known in the art.The results of this study will demonstrate that supplementation with aformulation of γ-tocopherol and LLU-α will treat and prevent theformation of neuropathological lesions associated with vitamin Edeficiency better than supplementation with α-tocopherol or nosupplementation at all.

MODULATION OF IMMUNE SYSTEM RESPONSE EXAMPLE 29

The main role of vitamin E in enhancing immune response is believed toinvolve the prevention of lipid perioxidation of cell membranes. Therapidly proliferating cells of the stimulated immune and phagocyticsystems are particularly prone to perioxidative damage by free radicals,peroxides, and superoxides. Vitamin E supplementation has been shown tomodulate the immune response of mammals as evidenced by a reduction inPGE₂ production, an increase in mitogenic response, an increase in IL-2production, and the induction of delayed-type hypersensitivity (DTH).(See Meydani and Tengerdy, Vitamin E and Immune Response, Chapter 40,Vitamin E in Health and Disease, Packer and Fuchs editors, Marcel DekkerInc. Publishers 1993 pp. 549-561). An improvement in immune responseafter supplementation with a formulation of γ-tocopherol and LLU-α aredetermined by measuring the reduction of PGE₂ and the increase in IL-2production in mice, according to the following example.

A first group of mice, the control group, does not receive treatmentwith γ-tocopherol or a formulation of γ-tocopherol and LLU-α. To comparethe therapeutic benefits of a formulation of γ-tocopherol and LLU-α withα-tocopherol, a control group which receives treatment with α-tocopherolis used. A second group of mice, the experimental group, receivestreatment with a formulation of γ-tocopherol and LLU-α. Treatmentconsists of 40 mg/kg of a formulation of γ-tocopherol 75% (weight toweight) and LLU-α 25% (weight to weight) for a period of 8 weeks.Shortly after the treatment phase, the control and experimental groupsare administered an antigen which illicits an immune response. Next, thePGE₂ production and IL-2 production is determined by conventionalmethods. The results of this study will demonstrate that mice whichreceived treatment with a formulation of γ-tocopherol and LLU-α exhibita lower level of PGE₂ and an increase in IL-2 production as compared tocontrol mice which received either α-tocopherol supplementation or nosupplementation at all.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims. All references cited herein are hereby expressly incorporated byreference.

What is claimed is:
 1. A medicament comprising more than 70%γ-tocopherol, wherein said medicament is in a unit dosage form suitablefor pharmaceutical administration.
 2. The medicament of claim 1, whereinthe amount of γ-tocopherol in said medicament is more than 75%.
 3. Themedicament of claim 1, wherein the amount of γ-tocopherol in saidmedicament is more than 80%.
 4. The medicament of claim 1, wherein theamount of γ-tocopherol in said medicament is more than 85%.
 5. Themedicament of claim 1, wherein the amount of γ-tocopherol in saidmedicament is more than 90%.
 6. The medicament of claim 1, wherein theamount of γ-tocopherol in said medicament is more than 95%.
 7. A methodof treating prostate cancer comprising administering a composition thatcomprises tocopherols, at least 50% of said tocopherols beingγ-tocopherol.
 8. The method of claim 7, wherein at least 55% of saidtocopherols are γ-tocopherol.
 9. The method of claim 7, wherein at least60% of said tocopherols are γ-tocopherol.
 10. The method of claim 7,wherein at least 75% of said tocopherols are γ-tocopherol.
 11. Themethod of claim 7, wherein at least 80% of said tocopherols areγ-tocopherol.
 12. The method of claim 7, wherein at least 85% of saidtocopherols are γ-tocopherol.
 13. The method of claim 7, wherein atleast 90% of said tocopherols are γ-tocopherol.
 14. The method of claim7, wherein at least 95% of said tocopherols are γ-tocopherol.
 15. Themethod of claim 7, wherein more than 95% of said tocopherols areγ-tocopherol.
 16. The medicament of claim 1, wherein said unit dosageform is a gelatin capsule.
 17. The medicament of claim 1, wherein saidunit dosage form comprises between 200 and 800 mg of γ-tocopherol. 18.The medicament of claim 16, wherein said geltin capsule comprisesbetween 200 and 800 mg of γ-tocopherol.